Project Summary Circadian clocks are endogenous ~24-hour oscillators that drive daily rhythms of biological processes. In mammals, circadian clocks are found in the brain and in most peripheral tissues. Together, the distributed clocks constitute the basic timing system that regulates physiology, metabolism, and behavior. The mammalian circadian clock is built on a transcriptional negative feedback loop that generates circadian rhythms at the molecular level. The transcription factor CLOCK-BMAL1 is at the heart of this feedback loop, acting as the core positive driver of circadian clock transcription. CLOCK-BMAL1 has been assumed to act in vivo as a heterodimer, but we have shown that in nuclear extracts from mammalian tissues it is exclusively embedded in an ~800-kDa protein complex during the transcriptional activation phase of the circadian cycle, far larger than the ~170-kDa heterodimer. Our preliminary biochemical, mass spectrometry, and single-particle electron microscopy results together unexpectedly suggest that this complex consists entirely or mostly of multiple CLOCK-BMAL1 heterodimers assembled into an oligomeric structure. These observations tell us that the full molecular nature and functional properties of the circadian clock transcriptional activator remain to be discovered, a fundamental gap in our understanding. The goal of this application is to analyze the composition, function, and three- dimensional structure of the enigmatic ~800-kDa CLOCK-BMAL1 complex. If successful, the project offers to extend and deepen our knowledge of circadian clock transcription, possibly to an atomic level of resolution. Advances in understanding the circadian clock will have important implications for our knowledge of the regulation of behavior and physiology, as well as for human health and disease. Circadian clock defects result in broad physiological dysfunction, producing disrupted sleep-wake cycles, metabolic syndrome, and increased cancer incidence. There are also links between clock dysfunction and mood disorders, including bipolar disorder. The proposed investigation aims to provide insights into the molecular machinery of the biological timing system essential for health.